Humanized antibodies against TL1A

ABSTRACT

Disclosed are humanized antibodies that bind specifically to TNF superfamily member 15 (TNFSF15), also known as TL1A. Methods of making and using the anti-TL1A antibodies are also described. The humanized antibodies may be antagonists and may used to treat or diagnose conditions associated with TL1A function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/585,792, filed Aug. 14, 2012, which is a Divisional of U.S. patentapplication Ser. No. 12/270,673, filed Nov. 13, 2008, now U.S. Pat. No.8,263,743, which claims the benefit of priority from U.S. ProvisionalPatent Application No. 60/987,651, filed on Nov. 13, 2007. The contentsof these applications are hereby incorporated by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-web and is herebyincorporated by reference in its entirety. Said ASCII copy, created onJul. 7, 2014, is named 075977-0279 SL.txt and is 72,106 bytes in size.

SUMMARY

The present invention is directed to antibodies against TL1A, andmethods of making and using such antibodies. The antibodies are expectedto be particularly useful in treating inflammatory conditions such asCrohn's disease.

BACKGROUND

Proteins that are structurally related to tumor necrosis factor (TNF)are collectively referred to as the TNF superfamily. TL1A, a TNFsuperfamily member, is a TNF-like cytokine that binds to thedeath-domain receptor (DR)3 and provides costimulatory signals toactivated lymphocytes. Through this interaction, TL1A induces secretionof IFN-gamma and may, therefore, participate in the development of Thelper-1-type effector responses.

TL1A is a type II transmembrane protein and has been designated TNFsuperfamily member 15 (TNFSF15). TL1A is expressed predominantly byendothelial cells and monocytes, and its expression is inducible byTNF-a and IL-1a. Migone et al., Immunity, 16:479-92 (2002). TL1a isupregulated by the proinflammatory cytokines TNF and IL-1 and also byimmune complexes (IC). Hsu et al., Exp. Cell Res., 292:241-51 (2004).

TL1A mediates signaling via its cognate receptor DR3, a death receptorwhose activation was known to induce both death and survival factors.TL1A, like TNF, is also presumed to circulate as a homotrimeric solubleform. Kim et al., J. Immunol. Methods, 298(1-2):1-8 (March 2005).

TL1A binds with high affinity to death receptor 3 (DR3) which is amember of the death-domain containing TNF receptor family, and is alsotermed Wsl-1, Apo-3, TRAMP, and LARD, and now designated TNF receptorsuperfamily member 25 (TNFRSF25). Depending on the cell context,ligation of DR3 by TL can trigger one of two signaling pathways,activation of the transcription factor NF-kB or activation of caspasesand apoptosis. TL1 functions in T cell costimulation and Th1polarization. On activated T cells, TL1A functions specifically via itssurface-bound receptor DR3 to promote cell survival and secretion ofproinflammatory cytokines. The secreted decoy receptor 3 (DcR3), asoluble protein of the tumor necrosis factor receptor (TNFR)superfamily, blocks the action of TL1A. Kim et al., “Identification ofnaturally secreted soluble form of TL1A, a TNF-like cytokine,” J ImmunolMethods, 298:1-8 (2005).

Potential Therapeutic Targets

Allergy and Asthma

Th2 polarization of CD4 T cells with elevated IgE levels and productionof IL-13 by NKT cells are major cause of lung inflammation in Allergyand asthma. TL1A plays a major role in allergic lung inflammation (Fanget al J. Exp. Med. 2008). TL co-stimulates IL-4 and IL-13 production inNKT cells. Blocking TL1A and DR3 interaction by TL1A antibody ordominant negative TL1A mutant abolishes lung inflammation.

Lung and Colon Carcinomas

Members in the TNF and its receptor superfamilies regulate immuneresponses and induce apoptosis. DR3 is preferentially expressed by Tlymphocytes and upregulated during T cell activation. The ligand for DR3is TL1A. TL1A also binds decoy receptor DcR3/TR6, which is expressed inseveral lung and colon carcinomas and in some normal tissues. TL1A isupregulated by proinflammatory cytokines TNF and IL-1. TL1A is a longervariant of TL1 (also called VEGI).

Atherosclerosis

In addition, TL1A has also been reported to be angiostatic and to inducemetalloproteinase and IL-8 gene expression (Su et al., Exp. Cell Res.,312:266-277 (2006); Kang et al., Cytokine, 29:229-235 (2005)). Indeed,TL1A and DR3 may be involved in the pathogenesis of atherosclerosis byincreasing the production of proinflammatory cytokines and chemokinesand decreasing plaque stability by inducing extracellularmatrix-degrading enzymes (Kang et al., Cytokine, 29:229-235 (2005)).

Rheumatoid Arthritis

There is also evidence to suggest that TL1A/DR3 is involved in theetiology of rheumatoid arthritis (Bossen et al., J. Biol. Chem.,281(20):13964-13971 (May 19, 2006).

Inflammatory Bowel Disease

Researchers have found an association of the expression of TL1A andinflammatory bowel disease (Prehn et al., Clin. Immunol., 112:66-77(2004); Bamias et al., J. Immunol., 171:4868-4874 (2003)).

Th1-Mediated Intestinal Diseases, Such as Crohn's Disease

Crohn's disease is a severe inflammatory bowel disorder that strikesyoung adults (ages 20-30). The condition is thought to originate frompredisposing genetic and environmental factors that cause an imbalanceof effector (proinflammatory) and regulatory T cell responses, resultingin inflammation of the gastrointestinal mucosa and disease.

The TL1A/DR3 pathway plays an important role in Th1-mediated intestinaldiseases, such as Crohn's disease. Konstantinos et al., The Journal ofImmunology, 2005, 174: 4985-4990 (2005); Bamias et al., J. Immunol.,171:4868-74 (2003). Blockade of the TL1A/DR3 pathway may, therefore,offer therapeutic opportunities in Crohn's disease.

TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 andIL-12/IL-18-stimulated peripheral blood (PB) T cells. Activation of DR3by TL induced the formation of a signaling complex containing TRADD,TRAF2, and RIP and activated the NF-kB and the ERK, JNK, and p38mitogen-activated protein kinase pathways. Kang et al., Cytokine,29:229-35 (2005). TL1A can be released to circulate as a homotrimericsoluble form. Wen et al., “TL1A-induced NF-kappaB activation and c-IAP2production prevent DR3-mediated apoptosis in TF-1 cells,” J. Biol.Chem., 278:39251-8 (2003).

Death receptors and their ligands play a key role in the maintenance oftissue homeostasis and the physiological regulation of programmed celldeath. Binding of a death ligand induces oligomerization of thereceptor, recruitment of an adapter protein via a conserved cytoplasmicsignaling element termed the death domain, activation of caspases, andinduction of apoptosis. Young et al., Proc Natl. Acad. Sci. USA.,103(22): 8303-8304 (May 30, 2006).

Although death receptors such as Fas/Apo-1/CD95, TNF-R1, TRAIL-R1,TRAIL-R2, or DR3 were initially characterized as inducers of apoptosis,there is growing evidence that these receptors also have nonapoptoticfunctions, including regulation of the adaptive immune response. Bamiaset al., Proc. Natl. Acad. Sci. USA, 103:8441-8446 (2006), report thatTL1A is expressed by lamina propia dendritic cells and that it functionsby increasing the proliferation of memory cells, but not naïve CD4⁺ Tcells, and synergizes with IL-12 and/or low-dose stimulation of the Tcell receptor to strongly enhance IFN-γ gene expression. IFN-γexpression in the gut has been considered a marker of inflammation, andmany strategies for treating Crohn's disease rely on broad attempts tosuppress the immune-activated state. However, such approaches (steroidtreatment and immunosuppressive drugs) do not focus on the gutspecifically and thus have their own complications. Targeted therapiesbased on the use of antagonists of TNF-α were introduced with success inthe 1990s, and the results reported in ref. 1 suggest that therapydirected specifically against TL1A or its receptor may provide analternative targeted therapy for this debilitating disorder.

As reported in Bamias et al., Proc. Natl. Acad. Sci. USA., 103:8441-8446(2006), TL1A seems to have a most profound effect when expressed in thegut during inflammation. TL1A synergizes in the induction of IFN-γexpression in human T cells when combined with IL-12/18, althoughincreased expression can also be observed in natural killer cells(Migone et al., Immunity., 16:479-492 (2002); Papadakis et al., J.Immunol., 174:4985-4990 (2005); Papadakis et al., J. Immunol.,172:7002-7007 (2004)). Bamias et al., Proc. Natl. Acad. Sci. USA.,103:8441-8446 (2006), is the first report of a similar observation inmouse models of Crohn's disease and extends earlier data by showing thatthe synergy occurs when the T cell receptor is weakly stimulated or Tcells are treated with IL-12. Although in Bamias et al. no synergy isobserved when TL1A treatment is combined with IL-18 this result may notbe surprising because both IL-18 and TL1A signal through NF-κB. Whereasthe initial report by Migone et al. on TL1A demonstrated that it was a Tcell costimulatory signal, Bamias et al. demonstrate that it is thememory T cell that most strongly responds, consistent with the increasedcapacity of this T cell population to express IFN-γ. Because thispopulation does proliferate, it also expresses higher levels of the TL1Areceptor, thus further enhancing the ability of the cells to proliferateand express IFN-γ. This finding might be considered somewhat surprisinggiven that the only known receptor of TL1A is DR3, a deathdomain-containing receptor, and it might have been hypothesized thattriggering this receptor would lead to cell death. (TL1A signals throughDR3, its only known cell surface receptor. TL1A also binds to thesoluble decoy receptor (DcR3)). However NF-κB-dependent antiapoptoticgenes, such as inhibitor of apoptosis 2, have been shown to be inducedby TL1A (Wen et al., J. Biol. Chem., 278:39251-39258 (2003)), andtherefore triggering of apoptosis vs. proliferation may be cell-typedependent.

Current treatment options for Crohn's disease include the monoclonalantibody against TNF-α infliximab (Remicade; Centocor, Inc., Horsham,Pa.), the monoclonal antibody Adalimumab (brand name Humira; Abbott), aswell as antiinflammatories (e.g., sulfasalazine), cortisone or steroids(e.g., prednisone), immune system suppressors (e.g. 6-mercaptopurine),and antibiotics. However, infliximab is the only treatment option havinga high degree of specificity; the remaining treatment options have a lowspecificity. Proc Natl Acad Sci U.S.A., 103(22): 8303-8304 (May 30,2006). This means that the treatment is not targeted to the diseasearea. While infliximab has a high specificity and is generally welltolerated, infliximab can cause recrudescence of tuberculosis infectionand worsening of heart failure, demyelinating disease, and an increasedincidence of lymphoma.

Therefore, there remains a need in the art for compositions that can beused in the treatment and diagnosis of diverse inflammatory and immunediseases and disorders, such as allergy/asthma, rheumatoid arthritis,multiple sclerosis, Crohn's disease, inflammatory bowel disease, chronicobstructive pulmonary disease, psoriasis, type 1 diabetes and transplantrejection. The present invention, directed to monoclonal antibodiesagainst TL1A, satisfies this need.

SUMMARY

Disclosed are antigen-binding polypeptide molecules that bindspecifically to the TNF-like cytokine TL1A (see GenBank accession no.AF520785). The polypeptides include a humanized heavy chain variableregion and a humanized light chain variable region. For example, thepolypeptides may include the framework (FR) regions of the light andheavy chain variable regions of a human antibody, while retainingsubstantially the antigen-binding specificity of a parental monoclonalantibody. The humanized heavy chain variable region and/or the humanizedlight chain variable region are at least about 87% humanized, at leastabout 90% humanized, at least about 95% humanized, at least about 98%humanized, or at least about 100% humanized, excluding thecomplementary-determining regions (CDRs). The antigen-bindingpolypeptides molecules may be derived from monoclonal antibody donors(e.g., mouse monoclonal antibody donors) and may include CDRs from themonoclonal antibodies (e.g., mouse monoclonal CDRs). The polypeptidesmay function as antagonists for the TL1A receptor.

Also encompassed by the invention are pharmaceutical compositionscomprising the polypeptides of the invention, methods of making suchpolypeptides and compositions, and methods of treating subjects in needwith the compositions of the invention. Exemplary conditions that may betreated with the compositions of the invention include, but are notlimited to autoimmune disease (e.g., lupus), inflammatory bowel disease(IBD), chronic obstructive pulmonary disease (COPD), arthritis (e.g.,rheumatoid arthritis), multiple sclerosis, transplant rejection, centralnervous system injury, Th1-mediated intestinal diseases such as Crohn'sdisease, psoriasis, leukemia or lymphoma (e.g., chronic lymphocyticleukemia (CLL)), atherosclerosis, and lung and colon carcinomas.

In some embodiments, the antigen-binding polypeptide binds specificallyto TL1A, and includes: (a) a humanized antibody heavy chain variableregion comprising: (1) a CDR-H1 comprising an amino acid sequence of({L,S,N}Y{G,A}MN) (SEQ ID NO: 1); (2) a CDR-H2 comprising an amino acidsequence of (WINT{Y,N}TG{E,N}PTYA{D,Q}{D,G}F{K,T}G) (SEQ ID NO: 2); and(3) a CDR-H3 comprising an amino acid sequence of(D{T,Y}{A,G}{M,K}{D,Y}{Y,G}{A,D}{M,Y}{A,Y}{Y,A}MDY) (SEQ ID NO: 3); and(b) a humanized antibody light chain variable region comprising: (1) aCDR-L1 comprising an amino acid sequence of({K,R}SSQ{N,S}{I,L}V{H,Y}S{D,N}GNTYL{E,N,D}) (SEQ ID NO: 4); (2) aCDR-L2 comprising an amino acid sequence of (KVSNR{F,D}S) (SEQ ID NO:5); and (3) a CDR-L3 comprising an amino acid sequence of({F,M}QG{S,T}H{V,-}{P,-}{T,-}) (SEQ ID NO: 6).

In certain embodiments the antigen-binding polypeptide bindsspecifically to TL1A and includes: a humanized antibody heavy chainvariable region comprising (1) the CDR-H1 comprising, consistingessentially of or consisting of the amino acid sequence of TSNMGVV (SEQID NO: 7); (2) the CDR-H2 comprising, consisting essentially of orconsisting of the amino acid sequence of HILWDDREYSNPALKS (SEQ ID NO:8); and (3) the CDR-H3 comprising, consisting essentially of orconsisting of the amino acid sequence of MSRNYYGSSYVMDY (SEQ ID NO: 9).

In some embodiments, the antigen-binding polypeptide comprises ahumanized antibody heavy chain variable region comprising, consistingessentially of or consisting of the amino acid sequence of:

(SEQ ID NO: 10) QVTLKESGPALVKPTQTLTLTCTFSGESLSTSNMGVVWIRQPPGKALEWLAHILWDDREYSNPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARMSRNYYGSSYVMDYWGQGTLVTVSS.

In some embodiments of the polypeptides, (1) the CDR-H1 consists of theamino acid sequence of (LYGMN) (SEQ ID NO: 11) or (NYGMN) (SEQ ID NO:12); (2) the CDR-H2 consists of the amino acid sequence of(WINTYTGEPTYADDFKG) (SEQ ID NO: 13); (3) the CDR-H3 consists of theamino acid sequence of (DTAMDYAMAY) (SEQ ID NO: 14) or DYGKYGDYYAMDY(SEQ ID NO: 15); (4) the CDR-L1 consists of the amino acid sequence of(KSSQNIVHSDGNTYLE) (SEQ ID NO: 16) or (RSSQSIVHSNGNTYLD) (SEQ ID NO:17); (5) the CDR-L2 consists of the amino acid sequence of (KVSNRFS) SEQID NO: 18); and (6) the CDR-L3 consists of the amino acid sequence of(FQGSHVPLT) (SEQ ID NO: 19).

In some embodiments, the polypeptide comprises a humanized antibodyheavy chain variable region of

(SEQ ID NO: 20) (Q{V,I}QLVQSG{S,P}ELKKPG{A,E}{S,T}VK{V,I}SCKASGYTFT{L,S,N}Y{G,A}MNWV{R,K}QAPG{Q,K}GL{E,K}WMGWINT{Y,N}TG{E,N}PTYA{D,Q}{D,G}F{K,T}GRF{V,A}FSL{D,E}TS{V,A}STAYLQI{S,N}{S,T}LK{A,N}ED{T,M}A{V,T}Y{Y,F}CARD{T,Y}{A,G}{M,K}{D,Y}{Y,G}{A,D}{M,Y}{A,Y}{Y,A}MDY)WGQGT{L,S}VTVSS).For example, the polypeptide may comprise a humanized antibody heavychain variable region of

(SEQ ID NO: 21) (QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDTAMDYAMAYWGQGTLVTVSS) or (SEQ ID NO: 22)(QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDTAMDYAMAYWGQGTLVTVSS).

Alternatively, the polypeptide may comprise a humanized antibody heavychain variable region of

(SEQ ID NO: 23) (QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYGKYGDYYAMDYWGQGTLVTVSS) or (SEQ ID NO: 24)(QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYGKYGDYYAMDYWGQGTLVTVSS).

In some embodiments, the polypeptide comprises a humanized antibodylight chain variable region of

(SEQ ID NO: 25) (DVVMTQ{T,S}PLSLPV{T,S}{P,L}G{E,D,Q}{P,Q}ASISC{K,R}SSQ{N,S}{I,L}V{H,Y}SDGNTYL{E,N}W{Y,F}{L,Q}Q{K,R}PGQSP{Q,K,R}{L,V,R}LIYKVSNR{F,D}SGVPDRFSGSGSGTDFTLKI{S,N}RVEAED{L,V}GVY{Y,F}C{F,M}QG{S,T}H{V,-}{P,-}{L,-}{T,-}{F,W}G{G,S,Q}GTK{V,L}EI KR).For example, the polypeptide may comprise a humanized antibody lightchain variable region of

(SEQ ID NO: 26) (DVVMTQTPLSLPVTPGEPASISCKSSQNIVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYY CFQGSHVPLTFGGGTKVEIKR)or (SEQ ID NO: 27) (DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPLTFGGGTKVEIKR).In another embodiment, the polypeptide may comprise a humanized antibodylight chain variable region of

(SEQ ID NO: 28) (DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPLTFGQGTKVEIKR).

Also disclosed are humanized antibody heavy chain variable regions. Thehumanized antibody heavy chain region may comprise: (1) a CDR-H1comprising an amino acid sequence of ({L,S,N}Y{G,A}MN) (SEQ ID NO: 29);(2) a CDR-H2 comprising an amino acid sequence of(WINT{Y,N}TG{E,N}PTYA{D,Q}{D,G}F{K,T}G) (SEQ ID NO: 2); and (3) a CDR-H3comprising an amino acid sequence of (DTAMDYAMAY) (SEQ ID NO: 14). Forexample, the humanized antibody heavy chain variable region may comprisean amino acid sequence of

(SEQ ID NO: 21) (QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDTAMDYAMAYWGQGTLVTVSS).Alternatively, the polypeptide may comprise a humanized antibody heavychain variable region of

(SEQ ID NO: 22) (QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDTAMDYAMAYWGQGTLVTVSS).

In another example, a humanized antibody heavy chain variable regioncomprises: (1) a CDR-H1 comprising an amino acid sequence of (NYGMN)(SEQ ID NO: 12); (2) a CDR-H2 comprising an amino acid sequence of(WINTYTGEPTYADDFKG) (SEQ ID NO: 13); and (3) a CDR-H3 comprising anamino acid sequence of (DYGKYGDYYAMDY) (SEQ ID NO: 15). For example, thehumanized antibody heavy chain variable region may comprise an aminoacid sequence of

(SEQ ID NO: 23) (QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYGKYGDYYAMDYWGQGTLVTVSS).Alternatively, the polypeptide may comprise a humanized antibody heavychain variable region of

(SEQ ID NO: 30) (QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYGKYGDYYAMDYWGQGTLVTVSS).

In another example, a humanized antibody heavy chain variable regioncomprises: (1) a CDR-H1 comprising an amino acid sequence of (NYAMS)(SEQ ID NO: 31); (2) a CDR-H2 comprising an amino acid sequence of(TIYSGGGYTFYLDSLKG) (SEQ ID NO: 32); and (3) a CDR-H3 comprising anamino acid sequence of (HSYPMTTVITYAPYYFYY) (SEQ ID NO: 33). Forexample, the humanized antibody heavy chain variable region may comprisean amino acid sequence of

(SEQ ID NO: 34) (QVQLVQSGSELKKPGASVKVSCKASGYTFTNYAMSWVKQAPGKGLKWMGTIYSGGGYTFYLDSLKGRFVFSLDTSVSTAYLQSSLKAEDTAVYFCARHSYPMTTVITYAPYYFYYWGQGTLVTVSS).

Also disclosed are humanized antibody light chain variable regions. Thehumanized antibody light chain variable region may comprise: (1) aCDR-L1 comprising an amino acid sequence of({K,R}SSQ{N,S}{I,L}V{H,Y}S{D,N}GNTYL{E,N,D}) (SEQ ID NO: 4); (2) aCDR-L2 comprising an amino acid sequence of (KVSNR{F,D}S) (SEQ ID NO:5); and (3) a CDR-L3 comprising an amino acid sequence of({F,M}QG{S,T}H{V,-}{P,-}{L,-}{T,-}) (SEQ ID NO: 6).

In other embodiments the antigen-binding polypeptide binds specificallyto TL1A and includes: a humanized antibody light chain variable regioncomprising: (1) a CDR-L1 comprising, consisting essentially of, orconsisting of an amino acid sequence of SASSSVNYMH (SEQ ID NO: 35); (2)a CDR-L2 comprising, consisting essentially of, or consisting of anamino acid sequence of STSNLAS (SEQ ID NO: 36); and (3) a CDR-L3comprising, consisting essentially of, or consisting of an amino acidsequence of HQWNNYGT (SEQ ID NO: 37).

In some embodiments, the antigen-binding polypeptide comprises ahumanized antibody light chain variable region comprising, consistingessentially of or consisting of the amino acid sequence of:

(SEQ ID NO: 38) DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWNN YGTEGQGTKVEIKR.

For example, the humanized antibody light chain variable region maycomprise an amino acid sequence of

(SEQ ID NO: 27) DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF QGSHVPLTFGGGTKVEIKR).In another embodiment, the polypeptide may comprise a humanized antibodylight chain variable region of

(SEQ ID NO: 28) (DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPLTFGQGTKVEIKR).Alternatively, the polypeptide may comprise a humanized antibody lightchain variable region of

(SEQ ID NO: 39) DVVMTQTPLSLPVTPGEPASISCKSSQNIVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCF QGSHVPLTFGGGTKVEIKR or(SEQ ID NO: 40) DVVMTQTPLSLPVSLGDQASISCKSSQNIVHSDGNTYLEWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDVGVYFCF QGSHVPLTFGGGTKLEIKR.

The humanized antibody light chain region may also comprise: (1) aCDR-L1 comprising an amino acid sequence of (RSSQSIVHSNGNTYLD) (SEQ IDNO: 17); (2) a CDR-L2 comprising an amino acid sequence of (KVSNRFS)(SEQ ID NO: 18); and (3) a CDR-L3 comprising an amino acid sequence of(FQGSHVPLT) (SEQ ID NO: 19). For example, the humanized antibody lightchain variable region may comprise an amino acid sequence of

(SEQ ID NO: 41) (DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLDWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPLTFGGGTKVEIKR.Alternatively, the polypeptide may comprise a humanized antibody lightchain variable region of

(SEQ ID NO: 42) (DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLDWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVY YCFQGSHVPLTFGQGTKVEIKR)).

In another example, a humanized antibody light chain variable regioncomprises: (1) a CDR-L1 comprising an amino acid sequence of(RSSQSIVHSNGNTYLD) (SEQ ID NO: 17); (2) a CDR-L2 comprising an aminoacid sequence of (KVSNRFS) (SEQ ID NO: 18); and (3) a CDR-L3 comprisingan amino acid sequence of (FQGSHVPLT) (SEQ ID NO: 19). For example, thehumanized antibody light chain variable region may comprise an aminoacid sequence of

(DVVMTQTPLSLPVTPGEPASISCRSSQSIVHSNGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGGGTKVEIKR) (SEQ ID NO: 43)) or(DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLDWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCFQGSHVPLTFGGGTKLEIKR) (SEQ ID NO: 44)).

The aforementioned humanized heavy chains and humanized light chains maybe present in the antigen binding polypeptides that binds specificallyto TL1A.

The antigen-binding polypeptide may be selected from the groupconsisting of an antibody molecule, a Fab fragment, a Fab′ fragment, aF(ab′)₂ fragment, and an scFv molecule. In some embodiments, thepolypeptide is an antibody molecule. Antibody molecules may includechimeric antibodies that include a human heavy chain constant region anda human light chain constant region. For example, the antibody moleculemay be an IgG molecule (e.g., a IgG1 or an IgG4 molecule), where thepolypeptide includes the heavy chain and light chain constant domains ofan IgG molecule. The polypeptide may be an scFv molecule. For example,the scFv may have a formula selected from the group consisting ofNH₂-L-VH-X-VK-COOH and NH₂-L-VK-X-VH-COOH; wherein L is a leadersequence; VH is the humanized antibody heavy chain variable region; X isa linking polypeptide; and VK is the humanized antibody light chainvariable region. The polypeptide may be an Fab HSA fusion molecule. Forexample, the Fab HSA fusion has a formula selected from the groupconsisting of NH₂-VH-CH1-HSA-COOH combined with NH₂-VK-CK-COOH; whereinthe VH-CH1-HSA is the humanized antibody heavy chain variable region(VH) and human constant heavy chain domain 1 (CH1) produced as a fusionprotein with human serum albumin (HSA) that then folds with its cognatehumanized antibody light chain variable region (VK) and human constantkappa domain (CK) to form the Fab HSA fusion protein.

The antigen-binding polypeptide further may be conjugated or fused to atherapeutic or diagnostic agent. For example, therapeutic agents may beselected from the group consisting of a cytotoxic agent, a radioactivelabel, an immunomodulator, a hormone, an enzyme, an oligonucleotide, aphotoactive therapeutic agent or a combination thereof. Examples ofdiagnostic agents may include a radioactive label, a photoactivediagnostic agent, an ultrasound-enhancing agent or a non-radioactivelabel.

The antigen-binding polypeptide may be an antagonist of TL1A. Typically,the polypeptide is not an agonist of TL1A.

The antigen-binding polypeptide binds to the TL1A receptor withspecificity and high affinity. Typically, the polypeptide binds to TL1Awith an affinity constant of at least about 10⁶M⁻¹ (preferably at leastabout 10⁷M⁻¹, more preferably at least about 10⁸M⁻¹, even morepreferably at least about 10⁹M⁻¹).

Also disclosed are pharmaceutical compositions comprising theaforementioned antigen-binding polypeptides and a carrier, such as adiluent or excipient. The pharmaceutical may further comprise anadditional therapeutic or diagnostic agent as disclosed herein.

Also disclosed are methods of treating or diagnosing a disease orcondition that comprise administering the disclosed pharmaceuticalcompositions to a patient in need thereof. For example, thepharmaceutical compositions may be administered to treat or diagnose aninflammatory, immune, and/or malignant disease or condition. Examples ofdiseases and conditions may include autoimmune disease (e.g., lupus),inflammatory bowel disease (IBD), chronic obstructive pulmonary disease(COPD), arthritis (e.g., rheumatoid arthritis), multiple sclerosis,transplant rejection, central nervous system injury, Crohn's disease,psoriasis, type 1 diabetes, lung and colon carcinomas, and leukemia orlymphoma (e.g., chronic lymphocytic leukemia (CLL)).

Also disclosed are polynucleotides that encode the aforementionedpolypeptides. The polynucleotides may be operably linked to a promoterfor expressing the encoded polypeptides in a suitable host cell. Assuch, methods of producing the polypeptide encoded by the recombinantpolynucleotide may include: a) culturing a cell transformed with therecombinant polynucleotide to express the encoded polypeptide; and b)recovering the polypeptide so expressed.

Both the foregoing general description and the following briefdescription of the drawings and detailed description are exemplary andexplanatory and are intended to provide further explanation of theinvention as claimed. Other objects, advantages, and novel features willbe readily apparent to those skilled in the art from the followingdetailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates inhibition of human TL1A (huTL1A)-induced Caspaseactivity on TF-1 cells by mouse and hamster anti-TL1A antibodies.Ab#1—19E06; Ab#2—12D01; Ab#3—15E09; Ab#4—16H02; Ab#5—14A03; Ab#6—04H08A;Ab#7—12F11; Ab#8—12D08.

FIG. 2 illustrates an alignment of the VH Domain of mouse anti-TL1A16H02 (SEQ ID NO: 70) with the closest human germline gene, IGHV7-4-1-02(SEQ ID NO: 69). The alignment was used as a template to create 2different versions of humanized 16H02 VH, identified in the figure asNew Hum 16H02 VH#1 (SEQ ID NO: 21) and VH#2 (SEQ ID NO; 22). FIG. 2discloses the majority sequences as SEQ ID NO; 68.

FIG. 3 illustrates the number of mutations from mouse to human andpercent humanization of two versions of a humanized anti-TL1A VH.

FIG. 4 illustrates an alignment of the VK domain of mouse anti-TL1A16H02 (SEQ ID NO: 74) with the closest human germline gene, A17 (SEQ IDNO: 73). The alignment was used as a template to create 2 differentversions of humanized 16H02 VH, identified in the figure as New Hum16H02 VK#1 (SEQ ID NO: 72) and VK#2 (SEQ ID NO: 26). FIG. 4 disclosesthe majority sequence as SEQ ID NO: 71.

FIG. 5 illustrates the number of mutations from mouse to human andpercent humanization of two versions of humanized anti-TL1A 16H02 VK.

FIG. 6 illustrates results of a transient transfection of 293F cellswith human 16H02 anti-TL1A VH#1 and VH#2 and human 16H02 VK#1 and VK#2to produce a full length humanized antibody molecule.

FIG. 7 illustrates the activity of 16H02 TL1A monoclonal antibodiesafter one round of humanization by showing the inhibition ofTL1A-induced caspase activity in TF-1 cells by the humanized antibodiescompared to mouse anti-TL1A antibody controls. 12D01=mouse anti-TL1anegative control; 16H02=mouse anti-TL1A positive control; 19E06=hamsteranti-TL1A control; 3009=humanized anti TL1A 16H02 VH#1+VK#1;3010=humanized anti TL1A 16H02 VH#2+VK#1; 3011=humanized anti TL1A 16H02VH#1+VK#2; 3012=humanized anti TL1A 16H02 VH#2+VK#2.

FIG. 8 illustrates the final mutations required for completehumanization of 16H02 VK framework. To create a fully humanized 16H02light chain the New Hum16H02 VK#2 (SEQ ID NO: 26) (see FIG. 4) startingsequence was aligned with the A17 germline sequence (SEQ ID NO: 76) and3 distinct regions or blocks identified (solid circles) for furthermutagenesis. Synthetic light chains were constructed that contained allpossible combinations of either the non-mutated wild type VK#2 sequence(=W) or A17 human germline sequence (=M) within each of the 3 regions orblocks. FIG. 8 discloses the majority sequence as SEQ ID NO: 75.

FIG. 9 illustrates the transient transfection ID and LDC # for finalversions of the humanized 16H02 VK. Synthetic light chains weregenerated that contained all possible combinations of either wild type(W) or mutant (M) sequence in 3 distinct regions or blocks of New Hum16H02 VK#2 from FIG. 8. The synthetic light chains were thencotransfected with New Hum 16H02 VH#1 from FIG. 2 and the resultingantibodies tested for inhibition of huTL1A-induced Caspase activity.

FIG. 10 illustrates inhibition of human TL1A (huTL1A)-induced Caspaseactivity on TF-1 cells by a panel of various humanized anti-TL1Aantibodies from FIG. 9. Activity of a fully humanized 16H02 TL1Aantibody identified as 3038 is compared to that of the original mouseanti-TL1A antibody 16H02.

FIG. 11 illustrates a sequence alignment of the VH Domain of five leadcandidate mouse anti-TL1A antibodies: 1B4 (SEQ ID NO: 58), 25B9 (SEQ IDNO: 59), 11D8 (SEQ ID NO: 60), 27A8 (SEQ ID NO: 61), and 38D6 (SEQ IDNO: 62).

FIG. 12 illustrates a sequence alignment of the VK Domain of five leadcandidate mouse anti-TL1A antibodies: 1B4 (SEQ ID NO: 49), 25B9 (SEQ IDNO: 50), 11D8 (SEQ ID NO: 52), 27A8 (SEQ ID NO: 51), and 38D6 (SEQ IDNO: 53).

FIG. 13 illustrates a sequence alignment of the humanized 1B4 VH domain(hum 1B4 VH AA) (SEQ ID NO: 77) with the original mouse VH domain (1B4VH AA) (SEQ ID NO: 58) and the closest matching human germline VH domain(VH2-70-10) (SEQ ID NO: 79.

FIG. 14 illustrates a sequence alignment of the humanized 1B4 VK domain(hum 1B4 VK AA) (SEQ ID NO: 67) with the original mouse VK domain (1B4VK AA) (SEQ ID NO: 49) and the closest matching human germline VK domain(VK-L8) (SEQ ID NO: 78).

FIG. 15 illustrates the inhibition of mammalian derived TL1A-inducedcaspase activity in TF-1 cells by humanized TL1A antibodies (1B4, 11D8,25B9) compared to the mouse 11D8 antibody.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Definitions

An antibody, as described herein, refers to a full-length (i.e.,naturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes) immunoglobulin molecule (e.g., an IgGantibody) or an immunologically active (i.e., specifically binding)portion of an immunoglobulin molecule, like an antibody fragment.

An antibody fragment is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, Fv, scFv and the like. Regardless of structure, anantibody fragment binds with the same antigen that is recognized by theintact antibody. The term “antibody fragment” includes aptamers,speigelmers, and diabodies. The term “antibody fragment” also includesany synthetic or genetically engineered protein that acts like anantibody by binding to a specific antigen to form a complex. Forexample, antibody fragments include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains, recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”), Fab HSA fusionpolypeptides in which the VH-CH1 are produced as a fusion to HSA, whichthen folds with its cognate VK-CK light chain to form a Fab, and minimalrecognition units consisting of the amino acid residues that mimic thehypervariable region.

A humanized antibody is a recombinant protein in which the CDRs from anantibody from one species, e.g., a rodent antibody, are transferred fromthe heavy and light variable chains of the rodent antibody into humanheavy and light variable domains or heavy and light variable domainsthat have been mutagenized to include at least a portion of the aminoacid sequence of the human heavy and light variable domains (asrepresented by “percent humanization”). The constant domains of theantibody molecule may be derived from those of a human antibody.

As used herein, “percent humanization” is calculated by determining thenumber of framework amino acid differences (i.e., non-CDR difference)between the humanized domain and the germline domain, subtracting thatnumber from the total number of amino acids, and then dividing that bythe total number of amino acids and multiplying by 100.

As used herein, “CDR” means a “complementarity determining region” thatis present in a variable domain of an antibody heavy chain (VH) or avariable domain of an antibody light chain (VL or VK). Each variabledomain includes three CDRs which are designated CDR-H1, CDR-H2, andCDR-H3, for those present in the heavy chain variable domain, andCDR-L1, CDR-L2, and CDR-L3 for those present in the light chain variabledomain. The Kabat numbering system is used herein. As such, CDR-H1begins at approximately amino acid 31 (i.e., approximately 9 residuesafter the first cysteine residue), includes approximately 5-7 aminoacids, and ends at the next tryptophan residue. CDR-H2 begins at thefifteenth residue after the end of CDR-H1, includes approximately 16-19amino acids, and ends at the next arginine or lysine residue. CDR-H3begins at approximately the thirty third amino acid residue after theend of CDR-H2; includes 3-25 amino acids; and ends at the sequenceW-G-X-G, where X is any amino acid. CDR-L1 begins at approximatelyresidue 24 (i.e., following a cysteine residue); includes approximately10-17 residues; and ends at the next tryptophan residue. CDR-L2 beginsat approximately the sixteenth residue after the end of CDR-L1 andincludes approximately 7 residues. CDR-L3 begins at approximately thethirty third residue after the end of CDR-L2 (i.e., following a cysteineresidue); includes approximately 7-11 residues and ends at the sequenceF or W-G-X-G, where X is any amino acid.

Conjugation to a Therapeutic or Diagnostic Agent

The antigen-binding polypeptides disclosed herein may be conjugated orfused to a therapeutic agent, which may include radioactive labels, animmunomodulator, a hormone, a photoactive therapeutic agent, a cytotoxicagent, which may be a drug or a toxin, and a combination thereof. Drugsmay include those drugs that possess the pharmaceutical propertyselected from the group consisting of antimitotic, antikinase,alkylating, antimetabolite, antibiotic, alkaloid, antiangiogenic,apoptotic agents and combinations thereof. More specifically, thesedrugs are selected from the group consisting of nitrogen mustards,ethylenimine derivatives, alkyl sulfonates, nitrosoureas, triazenes,folic acid analogs, COX-2 inhibitors, pyrimidine analogs, purineanalogs, antibiotics, enzymes, epipodophyllotoxins, platinumcoordination complexes, vinca alkaloids, substituted ureas, methylhydrazine derivatives, adrenocortical suppressants, antagonists,endostatin, taxols, camptothecins, anthracyclines, taxanes, and theiranalogs, and a combination thereof. The toxins encompassed by thepresent invention may be selected from the group consisting of ricin,abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, DNaseI, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin. Pseudomonas exotoxin, and Pseudomonas endotoxin.

Immunomodulators may be selected from the group consisting of acytokine, a stem cell growth factor, a lymphotoxin, a hematopoieticfactor, a colony stimulating factor (CSF), an interferon (IFN),erythropoietin, thrombopoietin and a combination thereof. Specificallyuseful are lymphotoxins such as tumor necrosis factor (TNF),hematopoietic factors, such as interleukin (IL), colony stimulatingfactor, such as granulocyte-colony stimulating factor (G-CSF) orgranulocyte macrophage-colony stimulating factor (GM-CSF)), interferon,such as interferons-alpha, -beta, or -gamma, and stem cell growthfactor, such as designated “S1 factor”. More specifically,immunomodulators may include IL-1, IL-2, IL-3, IL-6, IL-10, IL-12,IL-18, IL-21 interferon-gamma, TNF-alpha or a combination thereof.

The antigen-binding polypeptides disclosed herein may be conjugated orfused to a diagnostic agent. Diagnostic agents may include photoactivediagnostic agents or radiolabels having an energy between 60 and 4,000keV, or a non-radioactive label. The radioactive label is preferably agamma-, beta-, and positron-emitting isotope and is selected from thegroup consisting of ¹²⁵I, ¹³¹I, ¹²³I, ¹²⁴I, ⁸⁶Y, ¹⁸⁶Re, ¹⁸⁸Re, ⁶²Cu,⁶⁴Cu, ¹¹¹In, ⁶⁷Ga, ⁶⁸Ga, ^(99m)Tc, ^(94m)Tc, ¹⁸F, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Brand combinations thereof. Diagnostic agents may include contrast agents,for example, such as manganese, iron or gadolinium.

Exemplary Method of Making Anti-TL1A Antibodies Using HybridomaTechnology

BALB/c mice can be immunized with recombinant TL1A protein(extracellular domain). In a typical procedure 10 mg of protein in 50 mlof complete Freund's adjuvant (Sigma) is injected subcutaneously. Two tofour additional injections in incomplete Freund's adjuvant can be givenat 2 week intervals followed by a final boost in PBS. Alternatively,injections can be given in the foot pads. Three days later mice can besacrificed, their spleens or poplietal lymph nodes can be harvested andlymphocytes can be isolated for fusion. Lymphocytes can be fused withP3X63Ag8.653 plasmacytoma cells at 5:1 ratio using PEG/DMSO (Sigma) as afusion agent. After fusion cells can be resuspended in selective HATmedia and seeded at 10⁶ cells per well in 96 well plates. Thesupernatants from hybridomas that survived HAT selection can be screenedby direct binding ELISA for the presence of TL1A binding antibodies.Hybridomas secreting TL1A binding antibodies can be identified and theirsupernatants can be further screened by inhibition of binding ELISA forantibodies inhibiting binding of TL1A to its receptor DR3. Thehybridomas identified as positives for inhibition of TL1A binding canthen be screened for inhibition of TL1A induced caspase activity in TF-1cells to identify TL1A antagonistic clones.

Exemplary Antibody Humanization Strategy

One goal in humanizing the anti-TL1A antibodies is to obtain 70-100%humanized VH and VK domains that retain 90-100% of original bindingaffinity and specificity. Site-directed mutagenesis of individual highrisk positions in VH and VK can be used to further humanize theantibodies while maintaining binding affinity and specificity.

Humanization can be performed by CDR grafting and structure basedanalysis and variable region resurfacing. (See Jones et al., NATURE(1986) May 29-Jun. 4; 321(6069):522-5; Roguska et al., PROTEINENGINEERING, 1996, 9(10):895-904; and Studnicka et al., Humanizing MouseAntibody Frameworks While Preserving 3-D Structure. PROTEIN ENGINEERING,1994, Vol. 7, pg 805). The primary antibody sequence and 3-D structuredata can be utilized to identify key framework residues required tomaintain the binding affinity and specificity. The “Blast for Igsequences” website sponsored by the NCBI can be used to identify theclosest match to the mouse VH and VK region used in the study. Humangermline VH and VK genes can be chosen as the best matches to the mousesequence VH and VK sequences. Alternatively, sequences from thenaturally expressed human antibody repertoire can be used as a templatefor humanization either alone or in combination with the closestmatching human germ line gene.

After aligning mouse anti-TL1A VH and VK to the nearest human germlineor expressed repertoire of genes, the amino acid at every position canbe evaluated for potential influence on binding and immunogenicity. Thisinformation can be used to assign a low, moderate, or high risk valuefor mutation at each position. In one embodiment, only the low andmoderate risk positions are mutated while avoiding the high riskpositions. If necessary, an affinity maturation strategy can beperformed by incorporating tyrosines pair wise at each position in theCDR's of VH, VK or both.

Exemplary Cloning and Sequencing of Murine Anti-TL1A VH and VK Domainsfrom Hybridoma Cell Lines

Hybridoma cells can be pelleted, washed 3× with PBS and RNA extractedusing Trizol reagent (Invitrogen, Cat. No. 15596-026) following themanufacturers protocol. Total RNA can be converted to cDNA using a 5′RACE kit (Rapid Amplification of cDNA Ends, Invitrogen, Cat. No.18374-058) following the manufacturers protocol. Briefly, RNA can beligated to random hexamer primer, Random N6, and 1^(st) strand cDNA canbe generated using superscript II RNAase H negative reversetranscriptase. The cDNA can be purified using a GlassMax spin cartridgeprovided with the kit and then reacted with TdT (terminaldeoxynucleotidyl transferase) in the presence of dCTP to append the cDNAwith C basepairs at the 5′ end. The dC-tailed cDNA can be PCR amplifiedusing an anchor primer specific for the dC tail and a gene specificprimer that hybridizes to highly conserved DNA sequence in the mouseconstant heavy 1 (CH1) for VH and constant kappa (CK) for VK. Theresulting PCR product can be analyzed by gel electrophoresis for correctsize corresponding to intact VH or VK domain then purified and ligatedinto a TOPO TA vector (Invitrogen Cat. No. K4575-01) following themanufacturers protocol. After transformation into bacteria DNA can beprepared from clones containing the correct size insert and the DNAsequence can be determined using a Big Dye terminator sequencingreaction mix (Applied Biosystems, Part No. 4336699) and a 3700 ABI/PrismDNA analyzer following manufacturers protocol.

Exemplary Humanizing Murine Anti-TL1A Antibodies

Murine anti-TL1A antibodies can be identified based on binding data andsequence data generated as described above. The amino acid sequence ofthe VH and VK domains from these antibodies can be aligned to humangermline VH and VK domains using currently available public databases(i.e., Blast for IgG at the NCBI and V-base at the MRC). At thosepositions in the framework where the mouse sequence differed from thehuman germline, an iterative process can be used to convert or mutatethe mouse framework so it matches the corresponding human germlineframework. In addition, or alternatively, certain CDR amino acidresidues for both the VH and VK can be mutated by replacement withtyrosine (i.e., affinity matured) to potentially help compensate for anylosses in affinity due to the framework residues changes. The affinitymatured and humanized mouse VH and VK domains can be generated by apolymerase chain reaction process using a panel of overlapping syntheticDNA oligonucleotides. As part of the synthetic gene design process acodon optimization strategy can be used, that is to say the triplet codefor each amino acid that is preferentially utilized by mammalian cellsfor gene expression can be incorporated at each position. The syntheticVH and VK domains can be cloned into specialized mammalian expressionvectors that allow the corresponding domains to be expressed in thecontext of a fully human IgG1, G4 or Kappa antibody backbone.Small-scale production of the humanized antibodies can be achieved byco-transfection of an IgG1 or G4 construct with the Kappa construct into293F cells with lipofectamine (Invitrogen) following manufacturesprotocol. Supernatants from the transient transfections can be passedthrough Protein A or G resin and the IgG can be purified to homogeneityfor testing in cell based assays.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples. Allpublished and/or publicly available documents described herein arespecifically incorporated by reference.

Example 1

Amino Acid Sequences of VH and VK Domains of Mouse and Hamster anti-TL1Amonoclonal antibodies prepared as described herein are shown below. TheCDR regions of the variable domains are underlined.

12D08 VK (SEQ ID NO: 45): DVLMTQTPLS LPVSLGDQAS ISCRSSQSIV HSNGNTYLDW YLQKPGQSPN LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP LTFGAGTKLE LKR 16H02 VK (SEQ ID NO: 46):DVLMTQTPLS LPVSLGDQAS ISCKSSQNIV HSDGNTYLEW YLQKPGQSPK LLIYKVSNRF SGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YYCFQGSHVP LTFGSGTKLE IKR 15E09 VK (SEQ ID NO: 47):ETTVTQSPAS LSMAIGEKVT IRCITSTDID DDMNWYQQKP GEPPKLLISE GNTLRPGVPS RFSSSGYGTD FVFTIENMLS EDVADYYCLQ SDNLPLTFGA GTKLELKR 19E06 VK (SEQ ID NO: 48):DIVMTQSPSS LAVSTGGTVT LTCLSSQSLF SSDTNKNYLN WYLQKPGQSP KLLVYHASTR LTGVPDRFIG SGSGTDFTLT INSVQAEDLG DYYCQQHFRP PFTFGRGTKL EIKR IB4 VK AA  (SEQ ID NO: 49)QIVLTQSPAIMSASLGAEITLTC SASSSVNYMH WYQQRSGTSP KLLIY STSNLASGVPSRFSGSGSGTFYSLTISSVEAEDAADYY C HQWNNYGT FGGGTKLEIKR 25B9 VK AA (SEQ ID NO: 50) ENVLTQSPAILAASLGQKVTMTC SASSSVSSGYLH WYQQKSGA SPKPLIHRTSNLAS GVPPRFSGSGSGTSYSLSISSVEAEDDAT YYC QQWSGFPFT FGSGTKLEIKR27A8 VK AA  (SEQ ID NO: 51) DIVLTQSPASLTVSLGQRATISC RASQNVSTSSYSHMHWSQQK PGQPPKLLIK YASNLDS GVPARFSGSGSGTDFTLNIHPVEEED IATYYC QHSWEIPYTEGGGTKLHKR 11D8 VK AA  (SEQ ID NO: 52) DIVMTQSPASLTVSLGQRATISCRASQSVSTSSYSHMH WYQQK PGQPPKLLIR YASNLES GVPARFSGSGSGTDFTENIHPVEEEDTAIYYC QHSWELPYT EGGGTKLEIKR 38D6 VK AA  (SEQ ID NO: 53)DIVLTQFPASLPVSLGQRATISC RASQSVSTSSYSHMH WYQQK PGQPPKLLIT YASNLDSGVPARISGSGSGTDFTLNIHPVEEED TATYYC HHSWELPYT FGGGTKLEIKR12D08 VH (SEQ ID NO: 54): QIQLVQSGPE LKKPGETVKI SCKASGYTFT NYGMNWVKQA PGKGLKWMGW INTYTGEPTY ADDFKGRFAF SLETSASTAY LQINNLKNED MATYFCAKDY GKYGDYYAMD YWGQGTSVTV  SS16H02 VH (SEQ ID NO: 55): QIQLVQSGPE LKKPGETVKI SCKASGYTFT LYGMNWVKQA PGKGLKWMGW INTYTGEPTY ADDFKGRFAF SLETSASTAY LQINTLKNED MATYFCARDT AMDYAMAYWG QGTSVTVSS 15E09 VH (SEQ ID NO: 56):EVKLVDSGGG LVQPGDSLRL SCATSGFTFS DFYMEWVRQP PGKRLEWIAA SGNKANDYTT EYSASVKGRF IVSRDTSQSI LYLQMNDLRA EDTAIYYCVR DAGYGYWYFD VWGAGTTVTV  SS19E06 VH (SEQ ID NO: 57): QIQLQESGPS LVKPSQSLSL TCSVTGYSIT SDSYWNWIRQ FPGKNLVWMG YISYRGSTNY NPSLKSRISI TRDTSRNQFF LQLNSVTTED TATYYCARYS GYSFWYFDFW GQGTQVTVSS 1B4 VH  (SEQ ID NO: 58)a. QVTLKESGPGILQPSQTLSLTCSFSGFSLT TSNMGVV WIR QPSGKGLEWLLHILWDDREYSNPALKS RLTISKDPFNNQVFLK IANVDTADTATYYCAR MSRNYYGSSYVMDYWGQGTSVTVSS 25B9 VH  (SEQ ID NO: 59) EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMH WVKQKTGQ GLEWIG YINSNNDGTKYNEKFKG KATLTSDKSSSTAYMELSSLTSEDSAVYYCAT GDYYGGTSYWYFDV WGAGTTVTVSS 11D8 VH  (SEQ ID NO: 60)EVQLQQSGPELEKPGASVKISCKASGYSFT GYNMN WVKQSNGK SLEWIG NIDPYFGDTNYNQNFKGRATLTVDKSSNTAYMQLMSL TSEDSAVYYCAR EGAARAKNYFDY WGQGTTLTVSS 27A8 VH (SEQ ID NO: 61) EVQLQQSGPELETPGASVKISCKASGYSFTGYNMNWVKQTNGK SLEWIGNIDPYFGDANYNRKFKG KATLTVDKSSSTAYMQLRSL TSEDSAVYYCAK EGAARAKNYFDYWGQGTTLTVSS 38D6 VH AA  (SEQ ID NO: 62) EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMN WVRQTNGK SLEWIG HIDPYYGDATYRQKFKG KATLTVDKSSNTAYMQLKSLTSEDSAVYFCAR EGAARARNYFDY WGQGTTLTVSS

Example 2

This example describes an assay protocol to measure inhibition of TLA-induced caspase activity on TF-1 cells.

To determine neutralizing activity of anti-TL1A antibodies, theireffects on TL1A-induced caspase activity in TF-1 cells were determined.See FIG. 1. TF-1 cells were seeded at 75,000 cells/well in a black96-well plate with clear bottom in RPMI medium containing 1% fetalbovine serum. Cells were treated with 10 μg/mL cyclohexamide and 100ng/mL TL1A in the absence or presence of various concentrations of mouseor hamster parental TL1A antibodies for 6 hr at 37° C. Caspase activitywas measured by Apo-One homogeneous caspase-3/7 assay kit (Promega).Equal volume of Apo-One homogeneous caspase-3/7 assay buffer containingcaspase substrate, Z-DEVD-Rhodamine (SEQ ID NO: 63) was added to eachwell containing cells. After overnight incubation, fluorescence wasmeasured by a Wallac Victor2 fluorescence plate reader with excitationfilter 485 nm and emission filter 535 nm.

The results, shown in FIG. 1, shows that the level of fluorescence,which correlates with caspase activity, decreases with increasingconcentration of four (4) different anti-TL1A antibodies: Ab#1—19E06;Ab#3—15E09; Ab#4—16H02; and Ab#8—12D08.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

The invention claimed is:
 1. An isolated polynucleotide encoding anantigen-binding polypeptide that binds to specifically to TL1A,comprising: (a) a humanized heavy chain variable region comprising: (1)a CDR-H1 comprises the amino acid sequence selected from the groupconsisting of LYGMN (SEQ ID NO:11), NYGMN (SEQ ID NO:12), and TSNMGVV(SEQ ID NO:7); (2) a CDR-H2 comprises the amino acid sequence of(WINTYTGEPTYADDFKG) (SEQ ID NO:13) or HILWDDREYSNPALKS (SEQ ID NO:8);and (3) a CDR-H3 comprises the amino acid sequence selected from thegroup consisting of DTAMDYAMAY (SEQ ID NO:14), DYGKYGDYYAMDY (SEQ IDNO:15), and MSRNYYGSSYVMDY (SEQ ID NO:9); and/or (b) a humanized lightchain variable region comprising: (1) a CDR-L1 comprises the amino acidsequence selected from the group consisting of KSSQNIVHSDGNTYLE (SEQ IDNO:16), RSSQSIVHSNGNTYLD (SEQ ID NO:17) and SASSSVNYMH (SEQ ID NO:35);(2) a CDR-L2 comprises the amino acid sequence of KVSNRFS (SEQ ID NO:18)or STSNLAS (SEQ ID NO:36); and (3) a CDR-L3 comprises the amino acidsequence of FQGSHVPLT (SEQ ID NO:19) or HQWNNYGT (SEQ ID NO:37).
 2. Thepolynucleotide of claim 1, wherein the humanized heavy chain variableregion comprises the amino acid sequence of: (SEQ ID NO: 10)QVTLKESGPALVKPTQTLTLTCTFSGFSLSTSNNIGVVWIRQPPGKALEWLAHILWDDREYSNPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARMSRNYYGSSYVMDYWGQGTLVTVSS.


3. The polynucleotide of claim 1, wherein the humanized heavy chainvariable region comprises an amino acid sequence selected from the groupconsisting of: (a) (SEQ ID NO: 21)QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDTAMDYAMAYWGQGTLVTVSS; (b) (SEQ ID NO: 22)QVQLVQSGSELKKPGASVKVSCKASGYTFTLYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDTAMDYAMAYWGQGTLVTVSS; (c) (SEQ ID NO: 23)QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYGKYGDYYAMDYWGQGTLVTVSS; and (d) (SEQ ID NO: 24)QVQLVQSGSELKKPGASVKVSCKASGYTFTNYGMNWVRQAPGKGLKWMGWINTYTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARDYGKYGDYYAMDYWGQGTLVTVSS.


4. The polynucleotide of claim 1, wherein the humanized light chainvariable region comprises the amino acid sequence of: (SEQ ID NO: 38)DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWNNYGTFGQGT KVEIKR.


5. The polynucleotide of claim 1, wherein the humanized light chainvariable region comprises an amino acid sequence selected from the groupconsisting of (a) (SEQ ID NO: 27)DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGGGTKVEIKR; (b)(SEQ ID NO: 28) DVVMTQSPLSLPVTLGQPASISCKSSQNIVHSDGNTYLEWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGQGTKVEIKR; (c)(SEQ ID NO: 26) DVVMTQTPLSLPVTPGEPASISCKSSQNIVHSDGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGGGTKVEIKR; (d)(SEQ ID NO: 66) DVVMTQTPLSLPVSLGDQASISCKSSQNIVHSDGNTYLEWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGGGTKVEIKR; (e)(SEQ ID NO: 41) DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLDWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGGGTKVEIKR; (f)(SEQ ID NO: 42) DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLDWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPLTFGQGTKVEIKR; (g)(SEQ ID NO: 43) DVVMTQTPLSLPVTPGEPASISCRSSQSIVFISNGNTYLDWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQG SHVPLTFGGGTKVEIKR; and(h) (SEQ ID NO: 44) DVVMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLDWYLQKPGQSPKVLIYKVSNRFSGVPDRFSGSGSGTDFTLKINRVEAEDLGVYFCFQGSH VPLTFGGGTKLEIKR.


6. The polynucleotide of claim 1, wherein the polynucleotide encodes anantigen-binding polypeptide comprising: (a) a humanized heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of: (i) SEQ ID NO:10; (ii) SEQ ID NO:21; (iii) SEQ IDNO:22; (iv) SEQ ID NO:23; and (v) SEQ ID NO:24; and/or (b) a humanizedlight chain variable region comprising an amino acid sequence selectedfrom the group consisting of: (i) SEQ ID NO:38; (ii) SEQ ID NO:27; (iii)SEQ ID NO:28; (iv) SEQ ID NO:26; (v) SEQ ID NO:66; (vi) SEQ ID NO:41;(vii) SEQ ID NO:42; (viii) SEQ ID NO:43; and (ix) SEQ ID NO:44.
 7. Thepolynucleotide of claim 6, wherein the polynucleotide encodes anantigen-binding polypeptide comprising: (a) a humanized variable lightchain region comprising the amino acid sequence of: (SEQ ID NO: 38)DIQLTQSPSFLSASVGDRVTITCSASSSVNYMHWYQQKPGKAPKLLIYSTSNLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQWNNYG TFGQGTKVEIKR; and/or

(b) a humanized variable heavy chain region comprising the sequence of:(SEQ ID NO: 10) QVTLKESGPALVKPTQTLTLTCTFSGFSLSTSNMGVVWIRQPPGKALEWLAHILWDDREYSNPALKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARMSRNYYGSSYVMDYWGQGTLVTVSS.


8. The polynucleotide of claim 1, wherein the polynucleotide encodes anantigen-binding polypeptide selected from the group consisting of anantibody molecule, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment,and an scFv molecule.
 9. The polynucleotide of claim 8, wherein thepolypeptide is an antibody molecule.
 10. The polynucleotide of claim 9,wherein the antibody is a chimeric antibody comprising a humanized heavychain constant region and a humanized light chain constant region. 11.The polynucleotide of claim 9, wherein the antibody is an IgG molecule.12. The polynucleotide of claim 8, wherein the polypeptide is an scFvmolecule.
 13. The polynucleotide of claim 12, wherein the scFv moleculehas a formula selected from the group consisting of NH2-L-VH-X-VK-COOHand NH2-L-VK-X-VH-COOH, wherein: (a) L is a leader sequence; (b) VH isthe humanized antibody heavy chain variable region; (c) X is a linkingpolypeptide; and (d) VK is the humanized antibody light chain variableregion.
 14. The polynucleotide of claim 8, wherein the polypeptide is aFab HSA fusion molecule.
 15. The polynucleotide of claim 14, wherein theFab HSA fusion molecule has a formula selected from the group consistingof NH2-VH-CH1-HSA-COOH and NH2-HSA-CH1-VH-COOH combined withNH2-VK-CK-COOH, wherein VH-CH1-HSA or HSA-CH1-VH is the humanizedantibody heavy chain variable region (VH) and human constant heavy chaindomain 1 (CH1) produced as a fusion protein with human serum albumin(HSA) which folds with its cognate humanized antibody light chainvariable region (VK) and human constant kappa domain (CK) to form theFab-HSA or HSA-Fab fusion protein.
 16. The polynucleotide of claim 1,wherein the polynucleotide encodes an antigen-binding polypeptideconjugated to a therapeutic or diagnostic agent.
 17. The polynucleotideof claim 16, wherein: (a) the therapeutic agent is selected from thegroup consisting of a cytotoxic agent, a radioactive label, animmunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactivetherapeutic agent or a combination thereof; or (b) the diagnostic agentis selected from the group consisting of a radioactive label, aphotoactive diagnostic agent, an ultrasound-enhancing agent or anon-radioactive label.
 18. The polynucleotide of claim 1, wherein thepolypeptide binds to TL1A with an affinity constant of at least about106M-1.
 19. The polynucleotide of claim 1, wherein the polypeptide isencoded by cDNA.
 20. A recombinant polynucleotide comprising a promotersequence operably linked to the polynucleotides of claim
 1. 21. Anisolated cell transformed with the polynucleotide of claim
 20. 22. Amethod of producing the polypeptide encoded by the recombinantpolynucleotide of claim 20, the method comprising: (a) culturing a celltransformed with the recombinant polynucleotide to express the encodedpolypeptide; and (b) recovering the polypeptide so expressed.